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Updated: Jun 1, 2026
Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells
Published on: February 10, 2021
Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular
K Lueck1, S Wasmuth, J Williams
1Ophtha-Lab at Department of Ophthalmology, St Franziskus Hospital, Muenster, Germany.
Purpose:
There is evidence for complement dysfunction in age-related macular degeneration (AMD). Complement activation leads to formation of the membrane attack complex (MAC), known to assemble on retinal pigment epithelial (RPE) cells. Therefore, the effect of sub-lytic MAC on RPE cells was examined with regard to pro-inflammatory or pro-angiogenic mediators relevant in AMD.
Methods:
For sub-lytic MAC induction, RPE cells were incubated with an antiserum to complement regulatory protein CD59, followed by normal human serum (NHS) to induce 5% cell death, measured by a viability assay. MAC formation was evaluated by immunofluorescence and FACS analysis. Interleukin (IL)-6, -8, monocytic chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were quantified by enzyme-linked immunosorbent assay (ELISA). Intracellular MCP-1 was analysed by immunofluorescence, vitronectin by western blotting, and gelatinolytic matrix metalloproteinases (MMPs) by zymography.
Results:
Incubation of RPE cells with the CD59 antiserum followed by 5% NHS induced sub-lytic amounts of MAC, verified by FACS and immunofluorescence. This treatment stimulated the cells to release IL-6, -8, MCP-1, and VEGF. MCP-1 staining, production of vitronectin, and gelatinolytic MMPs were also elevated in response to sub-lytic MAC.
Conclusions:
MAC assembly on RPE cells increases the IL-6, -8, and MCP-1 production. Therefore, sub-lytic MAC might have a significant role in generating a pro-inflammatory microenvironment, contributing to the development of AMD. Enhanced vitronectin might be a protective mechanism against MAC deposition. In addition, the increased expression of gelatinolytic MMPs and pro-angiogenic VEGF may be associated with neovascular processes and late AMD.
Insights
Sub-lytic membrane attack complex (MAC) on retinal pigment epithelial cells promotes inflammation and may drive age-related macular degeneration (AMD). Enhanced vitronectin may offer protection, while MMPs and VEGF suggest neovascularization in late AMD.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Age-related macular degeneration (AMD) is linked to complement system dysfunction.
- The membrane attack complex (MAC), a product of complement activation, is found on retinal pigment epithelial (RPE) cells in AMD.
- Understanding MAC's effect on RPE cells is crucial for AMD pathogenesis.
Purpose of the Study:
- To investigate the impact of sub-lytic MAC on RPE cells.
- To determine if MAC induces pro-inflammatory and pro-angiogenic mediators relevant to AMD.
- To explore potential protective or pathological roles of molecules like vitronectin and MMPs.
Main Methods:
- Sub-lytic MAC was induced on RPE cells using anti-CD59 antiserum and normal human serum (NHS).
- MAC formation was confirmed via immunofluorescence and FACS analysis.
- Levels of IL-6, IL-8, MCP-1, and VEGF were quantified using ELISA; vitronectin and MMPs were also analyzed.
Main Results:
- Sub-lytic MAC was successfully induced on RPE cells.
- MAC stimulation led to increased release of IL-6, IL-8, MCP-1, and VEGF.
- Elevated intracellular MCP-1, vitronectin production, and gelatinolytic MMPs were observed.
Conclusions:
- Sub-lytic MAC on RPE cells enhances IL-6, IL-8, and MCP-1 production, potentially creating a pro-inflammatory environment in AMD.
- Increased vitronectin may act as a protective factor against MAC deposition.
- Elevated MMPs and VEGF suggest a role in neovascularization and advanced AMD stages.
